Methods And Interfaces For Single And Multidimentional Separations For Characterization And/Or Identification Of Molecules By Mass Spectrometry

ABSTRACT

The present invention relates a use of the electrocapture-based separation technology combined with mass spectrometry (e.g. sequence of polypeptides by collision-induce dissociation mass spectrometry, for the identification and/or characterization molecules of interest). In addition, it relates physical interfaces between electrocapture-based separations and different types mass spectrometers for on-line analysis, as well as the coupling of electrocapture-based separations, liquid chromatography and different types of mass spectrometrometers. It also relates the combination of the electrocapture-base separation technology with other liquid separation methods, as e.g. liquid chromatography, in order to achieve multidimensional separations prior mass spectrometrical analysis. The invention also relates to a separation device comprising a capture device, a fluidic connector e.g. an electrospray source, an electrospray interface-source and a mass spectrometer.

The present invention represents a novel mode to utilize a device described on PCT/SE2003/002027, WO 2004/056697 applications hereby enclosed. The key innovative steps in this invention are:

1. Methods and interfaces for the combination of Electrocapture-based separations (described on PCT/SE2003/002027, WO 2004/056697) with mass spectrometry for characterization and/or identification of molecules of interest. Mass spectrometry (MS) is a powerful analytical tool for identification and characterization of peptides, proteins, DNA, RNA, drugs and for other polymers and small molecules. Even though MS can analyze samples containing more that one particular type of molecules, a separation step is usually necessary when analyzing a sample with a complex mixture of molecules. This is particularly true for samples coming from biological sources as for example, blood, urine, saliva, cell extracts or fractions, bacteria extracts or fractions. Another important application where a separation step is necessary before mass spectrometrical analysis is in the identification of proteins via the enzyme digestion (e.g. trysin digestion) of a single protein (or a mixture of them) and the following separation and injection into the mass spectrometer. In this case peptides are separated and injected into the mass spectrometer, in which one peptide with a particular mass and charge ration (m/z) is selected for fragmentation followed by tandem mass spectrometry (MS/MS). Utilizing MS/MS, the m/z value of the fragment are determinated, thus making possible to determinate the amino acid sequence of the particular peptide, and therefore identify the protein from where the peptide was coming (via database search). For the later mixture of molecules, and the others mentioned above, the separation step improves the performance of overall analysis by mass spectrometry (higher number of molecules characterized and/or identified and increased sensitivity).

The connection of the electrocapture based separation with mass spectrometry is not an irrelevant issue, since molecules need to be ionized and in the gas-phase in order to be injected into the mass spectrometer.

The separation with the Electrocapture device must be performed in solution (molecules dissolved in a particular solvent), thus a particular interface and method should be developed to combine this two techniques.

In addition, another critical issue is that the interface (or connection) between both technologies must be done without destroy the separation process.

One of the innovative steps in this application is to combine electospray ionization mass spectrometry (ESI-MS) with the capture device to separate molecules of interest. In electrospray, molecules are ionized and transferred to the gas-phase by applying an electric field (about 1000 and 3000 kV) between the solution where the molecules of interest are dissolved and the mass spectrometer. All the aspects from the electrospray ionisation are not fully understood, but it is known that electrostatic-repulsion and solvent characteristics (evaporation, surface tension and pH) play an important role. In brief, the difference of electric potential between the solution and the mass spectrometer provokes the formation of the electrospray process, which involves the formation of micrometer and nanometer size droplets (due to an electrostatic effect) that have same charge. The latter causes that the droplets are repelled from each other (due to charge-to-charge repulsion). In parallel to this process, the solvent of the droplets starts to evaporate, which at the end, and together with the electrostatic repulsion make that the molecules are transferred to the gas-phase in an ionized state.

It is clear from the above that the electric potential between solvent and the mass spectrometer must be applied during the analysis of electrospray ionisation-mass spectrometry (ESI-MS). It is here where some problems arise from the connection of the capture device with ESI-MS, since the capture device has at least two electrodes by which the molecules are captured and separated. For this reason, the voltage from the ESI must not interferes with the voltage in the capture device (and vice-versa). Therefore, if proper voltage decoupling is not achieved, the electrospray process and/or the operation of the capture device will deteriorated to the extent that molecules are not separate/captured on the capture device and/or the electrospray process is not achieved.

The decoupling is achieved by the use of a capillary of a micrometer size dimensions (between 5 to 150 μm), and/or the use of buffers of low conductivity (using solutions of low salt concentration (e.g. between 1-20 mM and/or the use of solutions with organic solvents (e.g. acetonitrile, methanol) in a concentration between 99% to 1%) and/or the utilization of a capture device circuit voltage where the power supply of the capture device is electrically floating, thus does not interferes with the electrospray voltage. In addition, the decoupling can be done by using a sheath flow interface in which the electrospray voltage is applied to the electrospray solvent that travels coaxial to outlet of device, and is helped by a further coaxial flow of gas (sheath flow interfaces are generally known in the art and are for example described in the literature (e.g Electrophoresis 2004, 25, 1927-1948).

2. Another innovative step is the combination of electrocapture-based separations and chromatography separations with mass spectrometry. In addition to the connection of the capture device with electrospray ionization mass spectrometry, a further manner to increase the separation is to combine the electrocapture-based separation with a chromatography process (e.g. reverse phase chromatography) in order to carry out multidimensional separations, and by this means increase the separation power of the electrocapture-based separations, that will result in a improvement of performance of the mass spectrometric measurements (higher number of molecules characterized and/or identified and increased sensitivity). The most common manner to perform multidimensional separation before mass spectrometry is by combining ion-exchange chromatography (the separation is based on charge) with reverse-phase chromatography (the separation is based on hydrophobicity). The main problem with this approach is that the ion-exchange chromatography needs salts to separate the molecules of interest. Salts detrimental for the mass spectrometer, thus ion-exchange chromatography and reverse-phase chromatography can not be connected online, difficulting or hindering the automation of the overall separation procedure. The electrocapture-based separations is a method that separates molecules according to the electrophoretic mobility and does not use salts, making possible a straightforward connection to reverse-phase chromatography for multidimensional separation before mass spectrometry.

The invention will be described with the following figures of which

FIG. 1 Shows one device according to the invention. Reference FIG. 1 denotes a pump, 2 denotes fluidic conectors (pumpinjector-capturedevice), 3 denotes an injector, 4 denotes a capture device, 5 denotes an electrospray interface-source, 6 denotes a mass spectrometer, 7 denotes fluidic connector (capture device-electrospray source) and 8 denotes the inlet of the mass spectrometer.

FIG. 2 Shows details of the electrospray interface-source 5 of FIG. 1 wherein refrence FIG. 9 is a conductivity coated tip and reference FIG. 10 is a conector (zero or low dead volume).

FIG. 3. Shows on-line electrocapture-based Separations and ESI-Mass Spectra. The system setup consist in a 1 μL-injector, a syringe pump, a power supply, an Electrocapture device and a 50 μm fused silica capillary (20 cm) that connects the outlet of the Electrocapture device with the electrospray source. The source is a Silica capillary coated with a conductive material (for reference, see FIGS. 1 and 2), and the mass spectrometer is a Q-T of mass spectrometer. Peptides obtained from trypsin digestion of 4 proteins (BSA, myoglobin, ADH and cytochrome C) dissolved in 10 mM NH₄HCOO (pH 5,5 and 20% acetonitrile) are captured using an initial voltage drop of 300 V and a flow rate of 0.2 μL/min. As seen in the figure above, different peak profiles can be seen in the ESI-MS spectra by using different electrocapture voltages (200 and 250 V), proving evidence that the electrocapture device can be coupled online to ESI-MS to fractionate molecules of interest.

FIG. 4. Shows a sheath flow interface for the connection of the capture device with electrospray ionization mass spectrometry. The figure shows two differents manners to make the interface (A and B). The sheath flow interfaces allow decoupling between the capture device and electrospray voltages. In FIG. 4A the item 11 depicts a electrically conductive tube from which the electric field for the electrospray procces is applied. In conductive tube (11) a electrolyte solution is continously flowing (pumped). Item 12 depicts a tube where a gas is continously flowing through. The gas (sheath gas, item 14) and the electrolyte (sheath liquid, item 15) are travelling coaxial to a capillary tube (13) preferably made of silica, connected to fluidic connector (7). In FIG. 4B depict another setup to decouple the voltages, here, the item 11 depicts a electrically conductive tube from which the electric field for the electrospray procces is applied. In conductive tube (11) an electrolyte solution is continously flowing (pumped). Item 12 depicts a tube where a gas is continously flowing through. The gas and the electrolyte are travelling coaxial to the fluidic connector (7). The arrow shows the direction to mass spectrometer.

The invention also relates to a separation device comprising a capture device, a fluidic connector e.g. an electrospray source, an electrospray interface-source and a mass spectrometer. The electrospray interface-source may be a conductively coated tip connected to at least one conector. The conductive layer is made of any electrically conductive material such as a metal e.g. silver or gold. One or more chromatograhic columns separating by size or hydrophobicity could placed before or after the capture device.

All specifications regarding materials and performance apply mutated mutandis to both the methods and the devices according to the invention. 

1.-14. (canceled)
 15. An apparatus for analyzing molecules comprising: (a) an Electrocapture device comprising at least two electrodes, (b) an Electrospray source comprising a tip, (c) a mass spectrometer, (d) means to apply voltages to said electrodes, (e) means to apply an electric field between said Electrospray source and said mass spectrometer, and (f) An interface connecting said Electrocapture device and said Electrospray source.
 16. An apparatus according to claim 1, whereby said interface comprises a capillary tube.
 17. An apparatus according to claims 1 and 2, whereby said interface comprises a small bore capillary tube.
 18. An apparatus according to claims 1 and 2, whereby said interface comprises a fused silica capillary tube.
 19. An apparatus according to claims 1 and 2, whereby said interface comprises said capillary tube connected at its entrance end to said Electrocapture device and at its exit end to said Electrospray source.
 20. An apparatus according to claims 1 and 4, whereby said interface comprises said fused silica capillary tube connected at its entrance end to said Electrocapture device and at its exit end to said Electrospray source.
 21. An apparatus according to claims 1 through 6, whereby said interface comprises said capillary tube connects at its exit end to said tip coated with conductive material.
 22. An apparatus according to claims 1 through 7, whereby said interface comprises said tip is coated with conductive material.
 23. An apparatus according to claims 1 through 8, whereby said capillary tube exit end is connected to said tip with a zero or low or zero dead volume connection.
 24. An apparatus according to claim 1, whereby said interface comprises a sheath flow Electrospray interface.
 25. An apparatus according to claims 1 through 10, whereby said connection at said exit end of said capillary tube comprises a sheath flow interface with said Electrospray source.
 26. An apparatus according to claims 1, 10 and 11, whereby said sheath flow interface comprises a liquid sheath layer and a gas sheath layer flowing coaxially along said capillary tube.
 27. An apparatus according to claims 1, 10 and 11, whereby said sheath flow interface comprises a liquid sheath layer and a gas sheath layer flowing coaxially along said tip with said tip connected to said capillary tube.
 28. An apparatus according to claim 1, whereby said means to apply voltages to said electrodes comprises a power supply.
 29. An apparatus according to claims 1 and 14, whereby said power supply of said Electrocapture device is electrically floating.
 30. An apparatus according to claim 1, whereby said mass spectrometer comprises means to conduct mass to charge analysis of ions produced in said Electrospray source.
 31. An apparatus according to claim 1, whereby said mass spectrometer comprises means to conduct MS/MS analysis of ions produced in said Electrospray source.
 32. An apparatus according to claim 1, whereby said apparatus comprises a liquid chromatogram configured after said Electrocapture device.
 33. An apparatus according to claim 1, whereby said apparatus comprises a liquid chromatogram configured before said Electrocapture device.
 34. A method for analyzing molecules comprising: (a) utilizing an apparatus comprising an Electrocapture device with at least two electrodes, an Electrospray source comprising a tip, a mass spectrometer, means to apply voltages to said electrodes and means to apply an electric field between said Electrospray source and said mass spectrometer and an interface connecting said Electrocapture device and said Electrospray source, (b) conducting Electrocapture of said molecules in solution in said Electrocapture device and releasing of said molecules from said Electrocapture device, (c) Electrospraying said molecules released from said Electrocapture device forming gas phase ionized molecules, and (d) analyzing said ionized molecules using said mass spectrometer.
 35. A method according to claim 20, whereby said Electrocaptured molecules are separated during said releasing of said molecules from said Electrocapture device.
 36. A method according to claims 20 and 21, whereby said molecules are Electrocaptured and separated using said Electrocapture device with on line Electrospray ionization of separated molecules released from said Electrocapture device.
 37. A method according to claims 20, 21 and 22, whereby said separated molecules are mass to charge analyzed using said mass spectrometer.
 38. A method according to claims 20 through 23, whereby said separated molecules are MS/MS analyzed using said mass spectrometer.
 39. A method according to claims 20 through 24, whereby a liquid chromatography column is configured after said Electrocapture device and said molecules released from said Electrocapture device are further separated in said liquid chromatography column.
 40. A method according to claim 25 whereby two dimensional separation of said chemical species is conducted using said Electrocapture device and said liquid chromatography column on line with Electrospray MS and/or MS/MS analysis.
 41. A method according to claims 20 through 24, whereby a liquid chromatography column is configured before said Electrocapture device.
 42. A method according to claim 29, whereby said multidimensional separation is performed by first separating molecules in said liquid chromatography column and second by separating molecules in said Electrocapture device on line with Electrospray MS and/or MS/MS analysis.
 43. A method according to claims 20 through 28, whereby said liquid chromatography columns are configured before and after said Electrocapture device and multiple dimension separation of chemical species is conducted using said liquid chromatography columns and said Electrocapture device on line with Electrospray MS and/or MS/MS analysis.
 44. A method according to claim 20, whereby said Electrocapture of molecules is performed using buffers of low conductivity and/or using solutions with organic solvents.
 45. A method according to claim 30, whereby said low conductivity buffers comprise between 1 to 20 millimolar salt concentration and/or said organic solvent concentration comprises between 1% and 99% in water.
 46. A method according to claim 20, whereby said Electrospray is performed using sheath liquid and gas flow.
 47. A method according to claim 20, whereby said Electrospray is performed by spraying from a conductively coated tip.
 48. A method according to claim 20, whereby said decoupling is achieved by applying said voltages to said electrodes using an electrically floating power supply. 